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Antitumor Activity of Nitazoxanide against Colon Cancers: Molecular Docking and Experimental Studies Based on Wnt/β-Catenin Signaling Inhibition.

ABSTRACT: In colon cancer, wingless (Wnt)/β-catenin signaling is frequently upregulated; however, the creation of a molecular therapeutic agent targeting this pathway is still under investigation. This research aimed to study how nitazoxanide can affect Wnt/β-catenin signaling in colon cancer cells (HCT-116) and a mouse colon cancer model. Our study included 2 experiments; the first was to test the cytotoxic activity of nitazoxanide in an in vitro study on a colon cancer cell line (HCT-116) versus normal colon cells (FHC) and to highlight the proapoptotic effect by MTT assay, flow cytometry and real-time polymerase chain reaction (RT-PCR). The second experiment tested the in vivo cytotoxic effect of nitazoxanide against 1,2-dimethylhydrazine (DMH) prompted cancer in mice. Mice were grouped as saline, DMH control and DMH + nitazoxanide [100 or 200 mg per kg]. Colon levels of Wnt and β-catenin proteins were assessed by Western blotting while proliferation was measured via immunostaining for proliferating cell nuclear antigen (PCNA). Treating HCT-116 cells with nitazoxanide (inhibitory concentration 50 (IC50) = 11.07 µM) revealed that it has a more cytotoxic effect when compared to 5-flurouracil (IC50 = 11.36 µM). Moreover, it showed relatively high IC50 value (non-cytotoxic) against the normal colon cells. Nitazoxanide induced apoptosis by 15.86-fold compared to control and arrested the cell cycle. Furthermore, nitazoxanide upregulated proapoptotic proteins (P53 and BAX) and caspases but downregulated BCL-2. Nitazoxanide downregulated Wnt/β-catenin/glycogen synthase kinase-3β (GSK-3β) signaling and PCNA staining in the current mouse model. Hence, our findings highlighted the cytotoxic effect of nitazoxanide and pointed out the effect on Wnt/β-catenin/GSK-3β signaling.

SUBMITTER: Abd El-Fadeal NM

PROVIDER: S-EPMC8156814 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:PurposeThe T-cell-inflamed phenotype correlates with efficacy of immune-checkpoint blockade, whereas non-T-cell-inflamed tumors infrequently benefit. Tumor-intrinsic WNT/β-catenin signaling mediates immune exclusion in melanoma, but association with the non-T-cell-inflamed tumor microenvironment in other tumor types is not well understood.Experimental designUsing The Cancer Genome Atlas (TCGA), a T-cell-inflamed gene expression signature segregated samples within tumor types. Activation of WNT/β-catenin signaling was inferred using three approaches: somatic mutations or somatic copy number alterations (SCNA) in β-catenin signaling elements including CTNNB1, APC, APC2, AXIN1, and AXIN2; pathway prediction from RNA-sequencing gene expression; and inverse correlation of β-catenin protein levels with the T-cell-inflamed gene expression signature.ResultsAcross TCGA, 3,137/9,244 (33.9%) tumors were non-T-cell-inflamed, whereas 3,161/9,244 (34.2%) were T-cell-inflamed. Non-T-cell-inflamed tumors demonstrated significantly lower expression of T-cell inflammation genes relative to matched normal tissue, arguing for loss of a natural immune phenotype. Mutations of β-catenin signaling molecules in non-T-cell-inflamed tumors were enriched three-fold relative to T-cell-inflamed tumors. Across 31 tumors, 28 (90%) demonstrated activated β-catenin signaling in the non-T-cell-inflamed subset by at least one method. This included target molecule expression from somatic mutations and/or SCNAs of β-catenin signaling elements (19 tumors, 61%), pathway analysis (14 tumors, 45%), and increased β-catenin protein levels (20 tumors, 65%).ConclusionsActivation of tumor-intrinsic WNT/β-catenin signaling is enriched in non-T-cell-inflamed tumors. These data provide a strong rationale for development of pharmacologic inhibitors of this pathway with the aim of restoring immune cell infiltration and augmenting immunotherapy.See related commentary by Dangaj et al., p. 2943.

Project description:BackgroundChemotherapeutic agents that modulate cell cycle checkpoints and/or tumor-specific pathways have shown immense promise in preclinical and clinical studies aimed at anti-cancer therapy. MASTL (Greatwall in Xenopus and Drosophila), a serine/threonine kinase controls the final G2/M checkpoint and prevents premature entry of cells into mitosis. Recent studies suggest that MASTL expression is highly upregulated in cancer and confers resistance against chemotherapy. However, the role and mechanism/s of MASTL mediated regulation of tumorigenesis remains poorly understood.MethodsWe utilized a large patient cohort and mouse models of colon cancer as well as colon cancer cells to determine the role of Mastl and associated mechanism in colon cancer.ResultsHere, we show that MASTL expression increases in colon cancer across all cancer stages compared with normal colon tissue (P < 0.001). Also, increased levels of MASTL associated with high-risk of the disease and poor prognosis. Further, the shRNA silencing of MASTL expression in colon cancer cells induced cell cycle arrest and apoptosis in vitro and inhibited xenograft-tumor growth in vivo. Mechanistic analysis revealed that MASTL expression facilitates colon cancer progression by promoting the β-catenin/Wnt signaling, the key signaling pathway implicated in colon carcinogenesis, and up-regulating anti-apoptotic proteins, Bcl-xL and Survivin. Further studies where colorectal cancer (CRC) cells were subjected to 5-fluorouracil (5FU) treatment revealed a sharp increase in MASTL expression upon chemotherapy, along with increases in Bcl-xL and Survivin expression. Most notably, inhibition of MASTL in these cells induced chemosensitivity to 5FU with downregulation of Survivin and Bcl-xL expression.ConclusionOverall, our data shed light on the heretofore-undescribed mechanistic role of MASTL in key oncogenic signaling pathway/s to regulate colon cancer progression and chemo-resistance that would tremendously help to overcome drug resistance in colon cancer treatment.

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Antitumor Activity of Nitazoxanide against Colon Cancers: Molecular Docking and Experimental Studies Based on Wnt/β-Catenin Signaling Inhibition.

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